Current methods for detecting and/or quantifying nucleic acids of interest in clinical samples include nucleic acid amplification and real-time detection. See. e.g., U.S. Pat. Nos. 5,994,056 and 6,174,670 (measuring enhanced fluorescence of intercalating agents bound to double-stranded nucleic acids); and U.S. Pat. Nos. 5,455,175 and 6,174,670 (real time measurements carried out during the course of the reaction using a PCR cycler machine equipped with a fluorescence detection system and capillary tubes for the reactions). In these methods, as the amount of double-stranded material increases during amplification, the amount of signal also increases. Accordingly, the sensitivity of these systems depends upon a sufficient amount of double-stranded nucleic acid being produced to generate a signal that is distinguishable from background fluorescence. A variation of this system uses PCR primers modified with quenchers that reduce signal generation of fluorescent intercalators bound to a primer dimer molecule. See, e.g., U.S. Pat. No. 6,323,337.
Another method of detecting and/or quantifying nucleic acids of interest includes incorporation of fluorescent labels. See, e.g., U.S. Pat. No. 5,866,336. In this system, signal generation is dependent upon the incorporation of primers into double-stranded amplification products. The primers are designed such that they have extra sequences added onto their 5′ ends. In the absence of a complementary target molecule, the primers adopt stem-loop structures through intramolecular hybridization that bring a fluorescence resonance energy transfer (FRET) quencher into proximity with an energy donor, thereby preventing fluorescence. However, when a primer becomes incorporated into double-stranded amplicons, the quencher and donor are physically separated and the donor produces a fluorescent signal. The specificity of this system depends upon the specificity of the amplification reaction itself. Since the stem-loop sequences are derived from extra sequences, the Tm profile of signal generation is the same whether the amplicons were derived from the appropriate target molecules or from non-target sequences.
In addition to incorporation-based assays, probe-based systems can also be used for real-time analysis. For instance, a dual probe system can be used in a homogeneous assay to detect the presence of appropriate target sequences. In this method, one probe comprises an energy donor and the other probe comprises an energy acceptor. See European patent application publication no. 0 070 685. Thus, when the target sequence is present, the two probes can bind to adjacent sequences and energy transfer will take place. In the absence of target sequences, the probes remain unbound and no energy transfer takes place. Even if by chance there are non-target sequences in a sample that are sufficiently homologous that binding of one or both probes takes place, no signal is generated since energy transfer requires that both probes bind in a particular proximity to each other. See U.S. Pat. No. 6,174,670. The primer annealing step during each individual cycle can also allow the simultaneous binding of each probe to target sequences providing an assessment of the presence and amount of the target sequences. In a further refinement of this method, one of the primers comprises an energy transfer element and a single energy transfer probe is used. Labeled probes have also been used in conjunction with fluorescent intercalators, which combines the specificity of the probe methodology with the enhancement of fluorescence derived from binding to nucleic acids. See e.g., U.S. Pat. No. 4,868,103 and PCT Publication no. WO 99/28500.
Other types of probes used in real-time detection and/or quantification of nucleic acids of interest include an energy donor and an energy acceptor in the same nucleic acid. In assays employing these probes, the energy acceptor “quenches” fluorescent energy emission in the absence of complementary targets. See, e.g., U.S. Pat. No. 5,118,801 (“molecular beacons” used where the energy donor and the quencher are kept in proximity by secondary structures formed by internal base pairing). When target sequences are present, complementary sequences in the molecular beacons linearize by hybridizing to the target, thereby separating the donor and the acceptor such that the acceptor no longer quenches the emission of the donor, which produces signal. In Taqman, use is made of the double-stranded selectivity of the exonuclease activity of Taq polymerase. See U.S. Pat. No. 5,210,015. When target molecules are present, hybridization of the probe to complementary sequences converts the single-stranded probe into a substrate for the exonuclease. Degradation of the probe separates an energy transfer donor from the quencher, thereby releasing light from the donor. See U.S. Patent Publication no. 2005/0137388 (describing various formats for utilization of FRET interactions in various nucleic acid assays).
Probes comprising a non-fluorescent dark dye as energy acceptor (quencher) have also been used in the methods described above. When in close proximity, quenchers absorb emitted fluorescence from a donor dye and give no emission. Dabcyl is one such quencher with many applications, but its short absorption wavelength limits its use only to fluorescent reporters with short emission wavelengths, such as fluorescein and coumarin dyes. See, e.g., U.S. Pat. Nos. 5,866,336, 5,919,630, 5,925,517, and 6,150,097 and PCT publication nos. WO9513399A1, WO9929905A2, WO9949293A2, WO9963112A2. Dark quenchers that are suitable for pairing with long wavelength (red) fluorescent dyes have also been developed, but they generally have more complex structures, such as bisazo dyes (U.S. Pat. Nos. 7,019,129; 7,109,312; 7,582,432; 7,879,986; 8,410,255 and 8,440,399; and PCT publication no. WO2014021680), azo dyes containing nitro-substituted naphthalene moiety (U.S. Pat. Nos. 7,439,341 and 7,476,735), azo dyes containing 1,3,3-trimethyl-2-methyleneindoline ring system (U.S. Pat. No. 7,956,169), nitro-substituted non-fluorescent asymmetric cyanine dyes (U.S. Pat. Nos. 6,080,868 and 6,348,596), N-aryl substituted xanthene dyes (U.S. Pat. No. 6,323,337), dyes containing anthraquinone moieties (U.S. Pat. No. 7,504,495), and azo dyes containing heterocyclic moieties (US publication nos. 2010/0311184, and DE 102005050833 and DE 102005050834). Accordingly, there is a need for dark quencher dyes that have simple, non-complex structures that are able to absorb and quench fluorescence in a wider wavelength range.